Conventional cloning techniques rely upon T4 DNA ligase catalyzed ligation of DNA molecules with compatible cohesive or blunt termini. Ligation of cohesive termini is enabled by the formation of Watson-Crick base pairs between nucleotides present at the termini of the two molecules to be ligated. Ligation of molecules with blunt termini is less efficient, since base-pairing does not occur between the termini, and blunt end ligations therefore require higher concentrations of DNA and ligase.
There are several problems inherent with the use of T4 DNA ligase as a catalyst for DNA ligation: 1) T4 DNA ligase cannot catalyze the ligation of DNA molecules with incompatible termini. 2) There is a high frequency of intramolecular ligation when two or more species of DNA to be ligated contain compatible cohesive termini. To reduce the background or intramolecular ligation, it is first necessary to remove the 3' phosphate from one or more of the DNA species prior to ligation. 3) It is not possible to directionally ligate two species of DNA that contain compatible termini. 4) Site-specific ligation cannot occur when three or more species of DNA with compatible termini are present in the reaction.
There is a need for improved methods of DNA ligation. Applications of such improved method include cloning and polymerase chain reaction (PCR) protocols.
PCR is a powerful tool for producing multiple copies of a DNA molecules. Using PCR, it is possible to amplify DNA sequences to create thousands and millions of identical copies of a DNA molecule. PCR is thus used to clone DNA molecules from sources having at least a single copy of the sequence to be cloned.
PCR operates by providing primers, i.e. short single stranded polynucleotides which have sequences that are complementary to sequences of a portion of the nucleic acid molecule to be amplified. When PCR is performed, the primers and the DNA molecule to be amplified are combined and the temperature raised to denature the DNA molecule to be amplified into single stranded molecules. That is the double stranded DNA molecule dissociates into a sense strand and an antisense strand. The temperature is then lowered to promote hybridization of complementary sequences. Multiple copies of two primers are usually provided, one primer hybridizes to the sense strand of the sequence to be amplified and one primer hybridizes to the antisense strand. Using a thermostable polymerase and free nucleotides, a nucleotide molecule complementary to the sense strand is assembled by adding nucleotides to the 3' end of primer that is hybridized to the sense strand. Each free nucleotide added is complementary to the nucleotide on the sequence to be amplified. As the polymerization continues, a single stranded polynucleotide molecule is assembled nucleotide by nucleotide to be complementary to the sense strand of the sequence to be amplified starting from the 3' end of the primer and proceeding in the direction 5' to 3'. Simultaneously, using polymerase and free nucleotides, a nucleotide molecule complementary to the antisense strand is assembled by adding nucleotides to the 3' end of primer that is hybridized to the antisense strand. Each free nucleotide added is complementary to the nucleotide on the sequence to be amplified. As the polymerization continues, a single stranded polynucleotide molecule is assembled nucleotide by nucleotide to be complementary to the antisense strand of the sequence to be amplified starting from the 3' end of the primer and proceeding in the direction 5' to 3'. The temperature is then raised to dissociate hybridized complementary sequences after which the temperature is again lowered to promote hybridization. The primers hybridize to the original DNA molecule as well as to the molecules synthesized in the original polymerization. Once hybridized, the polymerase assembles the primers and free nucleotides into a DNA molecule which has a full length complementary sequence to the molecule that the primer is hybridized to. After numerous rounds of lowering temperature, hybridizing primers to molecules, formation of sequences complementary to the molecules by polymerization, raising the temperature to dissociate hybridized and repeating the hybridization/polymerization cycles, most of the amplification products are molecules with sequences identical to the sequence of the original molecule between the two primers.
One shortcoming of PCR is that there is a limit to how long a sequence can be amplified using the technology. If a sequence is greater than the limit for PCR can effectively be used for amplification, it must be amplified as a series of PCR products representing adjacent portions of the final desired molecules. The series of PCR products are ligated together to form the final desired molecules.
There is a need for compositions and methods for amplifying DNA molecules that have sequences which exceed the limit beyond which PCR is effective. There is a need for compositions and improved methods for ligating adjacent PCR products. There is a need for compositions and improved methods for ligating non-adjacent PCR products into one contiguous molecule.